Systems biology approach for enhancing limonene yield by re-engineering Escherichia coli

• Published in: NPJ systems biology and applications Vol. 10; no. 1; pp. 109 - 14
• Main Authors: Khanijou, Jasmeet Kaur, Hee, Yan Ting, Scipion, Clement P. M., Chen, Xixian, Selvarajoo, Kumar
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.10.2024; Nature Publishing Group; Nature Portfolio
• Subjects: 631/553/2695; 631/553/318; Alcohol dehydrogenase; Aldehyde dehydrogenase; Aldehyde Dehydrogenase - genetics; Aldehyde Dehydrogenase - metabolism; Bioinformatics; Biology; Biomedical and Life Sciences; Computational Biology/Bioinformatics; Computer Appl. in Life Sciences; Computer Simulation; Dehydrogenases; E coli; Enzymes; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Hexokinase; L-Lactate dehydrogenase; Life Sciences; Limonene; Limonene - metabolism; Metabolic Engineering - methods; Metabolic networks; Metabolic Networks and Pathways - genetics; Metabolomics; Metabolomics - methods; Mevalonate pathway; Mevalonic Acid - metabolism; Models, Biological; Systems Biology; Systems Biology - methods; Terpenes - metabolism
• ISSN: 2056-7189; 2056-7189
• DOI: 10.1038/s41540-024-00440-7

Engineered microorganisms have emerged as viable alternatives for limonene production. However, issues such as low enzyme abundance or activities, and regulatory feedback/forward inhibition may reduce yields. To understand the underlying metabolism, we adopted a systems biology approach for an engineered limonene-producing Escherichia coli strain K-12 MG1655. Firstly, we generated time-series metabolomics data and, secondly, developed a dynamic model based on enzyme dynamics to track the native metabolic networks and the engineered mevalonate pathway. After several iterations of model fitting with experimental profiles, which also included 13 C-tracer studies, we performed in silico knockouts (KOs) of all enzymes to identify bottleneck(s) for optimal limonene yields. The simulations indicated that ALDH/ADH (aldehyde dehydrogenase/alcohol dehydrogenase) and LDH (lactate dehydrogenase) suppression, and HK (hexokinase) enhancement would increase limonene yields. Experimental confirmation was achieved, where ALDH-ADH and LDH KOs, and HK overexpression improved limonene yield by 8- to 11-fold. Our systems biology approach can guide microbial strain re-engineering for optimal target production.